The invention relates generally to systems and methods for analyzing a sample for the presence of one or more nucleic acids, and more particularly, to systems and methods for conducting multi-stage nucleic acid amplification reactions, especially polymerase chain reactions (PCRs), under closed conditions.
Nucleic acid amplification reactions are crucial for many research, medical, and industrial applications. Such reactions are used in clinical and biological research, detection and monitoring of infectious diseases, detection of mutations, detection of cancer markers, environmental monitoring, genetic identification, detection of pathogens in biodefense applications, and the like, e.g. Schweitzer et al., Current Opinion in Biotechnology, 12: 21-27 (2001); Koch, Nature Reviews Drug Discovery, 3: 749-761 (2004). In particular, polymerase chain reactions (PCRs) have found applications in all of these areas, including applications for viral and bacterial detection, viral load monitoring, detection of rare and/or difficult-to-culture pathogens, rapid detection of bio-terror threats, detection of minimal residual disease in cancer patients, food pathogen testing, blood supply screening, and the like, e.g. Mackay, Clin. Microbiol. Infect., 10: 190-212 (2004); Bernard et al., Clinical Chemistry, 48: 1178-1185 (2002). In regard to PCR, key reasons for such widespread use are its speed and ease of use (typically performed within a few hours using standardized kits and relatively simple and low cost instruments), its sensitivity (often a few tens of copies of a target sequence in a sample can be detected), and its robustness (poor quality samples or preserved samples, such as forensic samples or fixed tissue samples are readily analyzed), Strachan and Read, Human Molecular Genetics 2 (John Wiley & Sons, New York, 1999).
Despite the advances in nucleic acid amplification techniques that are reflected in such widespread applications, there is still a need for further improvements in speed and sensitivity, particularly in such areas as infectious disease detection, minimum residual disease detection, bio-defense applications, and the like.
Significant improvements in sensitivity of PCRs have been obtained by using nested sets of primers in a two-stage amplification reaction, e.g. Albert et al., J. Clin. Microbiol., 28: 1560-1564 (1990). In this approach, the amplicon of a first amplification reaction becomes the sample for a second amplification reaction using a new set of primers, at least one of which binds to an interior location of the first amplicon. While increasing sensitivity, the approach suffers from increased reagent handling and increased risk of introducing contaminating sequences, which can lead to false positives. Attempts have been made to overcome these obstacles with so-called closed-tube nested PCRs; however, such approaches rely primarily on schemes for sequestering reagents in different sections of the same reaction vessel such that a second-stage reaction may be initiated by forcing reagents together by some physical process, such as centrifugation, e.g. Youmo, PCR Methods and Applications, 2: 60-65 (1992); Wolff et al., PCR Methods and Applications, 4: 376-379 (1995); Olmos et al., Nucleic Acids Research, 27: 1564-1565 (1999). Thus, substantial portions of first-stage reaction components are present in the second-stage reaction.
Significant improvements in sensitivity and a reduction of false positives have also been obtained by carrying out reactions in closed environments. A drawback of highly sensitive amplification techniques is the occurrence of false-positive test results, caused by inappropriate amplification of non-target sequences, e.g. Borst et al., Eur. J. Clin. Microbiol. Infect. Dis., 23: 289-299 (2004). The presence of non-target sequences may be due to lack of specificity in the reaction, or to contamination from prior reactions (i.e. “carry over” contamination) or to contamination from the immediate environment, e.g. water, disposables, reagents, etc. Such problems can be ameliorated by carrying out amplifications in closed vessels, so that once a sample and reagents are added and the vessel sealed, no further handling of reactants or products takes place. Such operations have been made possible largely by the advent of “real-time” amplifications that employ labels that continuously report the amount of a product in a reaction mixture.
Despite the attempts at multi-stage amplifications in closed vessels, the current art lacks methods or systems in which multi-stage reactions can take place without the possibility of there being interfering effects from undesired components, e.g. primers or other components, of prior reactions. Accordingly, there remains a need for new approaches for carrying out closed multi-stage amplification reactions that have the convenience of single-stage techniques, but which have the greater sensitivity afforded by a multi-stage amplification using nested primers.